PROJECT SUMMARY Nontuberculous mycobacteria (NTM) have emerged as an increasingly common cause of severe lung disease in patients with cystic fibrosis (CF). Unfortunately, diagnosis, prognosis, and treatment remain exceedingly difficult. Infection can result in more rapid decline in lung function and even death. During chronic infection, NTM frequently become resistant to the most effective antibiotic regimens, and infection is typically difficult or impossible to eradicate. Moreover, little is known about the molecular determinants of pathogenesis, inflammation, immune evasion, and persistence in NTM-driven disease. Here we propose an approach that follows the genomic evolution of NTM over time in chronically infected CF patients. We will rigorously apply whole genome sequencing and a battery of phylogenomic analyses to identify genetic changes in NTM strains that are likely to increase pathogen fitness. Because the CF lung harbors a complex and dynamic microbial ecosystem we will also comprehensively assess co-inhabiting microbiota, including well-known CF pathogens and other members of the respiratory microbiota. While some microbiota may compete with NTM, others may act to reinforce NTM infections, and some could even serve as reservoirs for genes that are beneficial to the pathogen (e.g., antibiotic resistance). We hypothesize that NTM undergoes predictable phenotypic and genotypic changes, which are likely influenced by co-infecting microbiota, to allow for enhanced NTM fitness, virulence, and persistence in the lung. AIM 1 examines longitudinal isolates from CF patients infected with NTM, emphasizing a deep sampling approach and population-level estimates of microbial diversity, mutation, recombination, and natural selection. Evolutionary changes in NTM populations will be associated with detailed clinical and phenotypic information, to identify genes that may be involved in antibiotic resistance, virulence, and persistence. AIM 2 uses a novel, whole 16S-rRNA gene sequencing approach to examine the cross-sectional and longitudinal interaction of NTM with other lung microbiota including co- infecting CF pathogens. We will compare patients with and without NTM and associate differences in the microbial population. Completing these aims will identify genes and microbial interactions that can be targeted for future functional studies aimed at uncovering the basic mechanisms and strategies that allow NTM adaptation during in-host evolution. We will also test and validate bioinformatic pipelines and sampling strategies that can be used in future NTM phylogenomic studies, and we will gain a fine-grained understanding of the NTM population dynamics during chronic infection. Characterizing in-host NTM adaptation will allow us to more effectively identify strains that may be difficult to eradicate or treat, and design new, targeted interventions to subvert these adaptations.!